At the indicated time points, cells were fixed with 3.7% paraformaldehyde (PFA) and permeablized with 0.1% Triton X-100 prior to immunocytochemistry. Anti-vinculin and anti-paxillin antibodies (Sigma-Aldrich, St. Louis, MO) were added and incubated at 4°C overnight. The following day, appropriate secondary antibodies (AlexaFluor 647-labeled, phalloidin-AlexaFluor 488, and DAPI - all from Life Technologies/Invitrogen, Grand Island, NY) were added. Actin was also visualized in TCPS- and scaffold-attached cells after transfection with a BacMam actin-GFP transduction reagent (Life Technologies/Invitrogen), according to the manufacturer’s instructions.
Additionally, coverslip-attached scaffolds containing live cells were incubated with PKH26 (Sigma-Aldrich), a hydrophobic red fluorescent label [28 (link)]. The scaffolds were mounted in Fluoromount™ (Sigma-Aldrich) and imaged using Olympus Filter FV1000 and Olympus Spectral FV1000 confocal systems (Olympus America Inc., Melville, NY). Images were viewed using the Olympus FV10-ASW software.
Additionally, coverslip-attached scaffolds containing live cells were incubated with PKH26 (Sigma-Aldrich), a hydrophobic red fluorescent label [28 (link)]. The scaffolds were mounted in Fluoromount™ (Sigma-Aldrich) and imaged using Olympus Filter FV1000 and Olympus Spectral FV1000 confocal systems (Olympus America Inc., Melville, NY). Images were viewed using the Olympus FV10-ASW software.